Potent Phosphodiesterase Inhibitor Research Articles

Characterization and effects of methyl-2- (4-aminophenyl)-1,2-dihydro-1-oxo-7- (2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032), a novel potent inhibitor of cGMP-binding cGMP-specific phosphodiesterase (PDE5)

An isoquinolone derivative, methyl-2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032), was found to be a novel potent inhibitor of cyclic GMP (cGMP)-binding cGMP-specific phosphodiesterase (PDE5). We investigated the inhibitory effects of T-1032 on six PDE isozymes isolated from canine tissues. T-1032 specifically inhibited the hydrolysis of cGMP by PDE5 partially purified from canine lung, at a low concentration ( ic 50 = 1.0 nM, K i = 1.2 nM), in a competitive manner. In contrast, the ic 50 values of T-1032 for PDE1, PDE2, PDE3, and PDE4 were more than 1 μM. T-1032 also inhibited PDE6 from canine retina with an ic 50 of 28 nM, which is of the same order of magnitude as the ic 50 of sildenafil. cGMP hydrolytic activities of two alternative splice variants of canine PDE5 expressed in COS-7 cells were inhibited by this compound to a similar extent. T-1032 increased the intracellular concentration of cGMP in cultured rat vascular smooth muscle cells in the presence and absence of C-type natriuretic peptide, an activator of membrane-bound guanylate cyclase, whereas the compound did not change cyclic AMP levels. These data indicated that T-1032, which belongs to a new structural class of PDE5 inhibitors, is a potent and selective PDE5 inhibitor. This compound may be useful in pharmacological studies to examine the role of a cGMP/PDE5 pathway in tissues.

CAMP-dependent protein kinase control of plasma membrane lipid architecture in boar sperm.

Bicarbonate/CO(2), a physiological effector of sperm capacitation, has been shown to induce a rapid and reversible change in the lipid architecture of the plasma membrane of live boar sperm: the change is detectable as an increase in the cells' ability to bind the fluorescent dye merocyanine, a characteristic which implied an increase in lipid packing disorder (Harrison et al. 1996. Mol Reprod Dev 45:378-391). Evidence suggested that cAMP may act as a second messenger in the system, and we have therefore investigated this cAMP-dependency in more detail. Bicarbonate stimulates cAMP levels within 1 min in a dose-dependent fashion, prior to parallel increases in merocyanine binding. Although the potent somatic cell adenylyl cyclase activator forskolin is unable to induce significant increases in cAMP or merocyanine binding, increases in merocyanine binding are inducible in a dose-dependent fashion by 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole 3',5'-cyclic monophosphothioate, a cAMP analogue highly specific in its ability to stimulate protein kinase A; moreover, the bicarbonate-induced membrane change is inhibited by H89, a specific protein kinase A inhibitor. Neither bisindolylmaleimide I (protein kinase C inhibitor) nor lavendustin A (protein tyrosine kinase inhibitor) are inhibitory. In the presence of low levels of the potent phosphodiesterase inhibitor papaverine, increases in merocyanine binding are enhanced by okadaic acid and (more effectively) by calyculin (both protein phosphatase inhibitors). We conclude that boar sperm plasma membrane lipid architecture is controlled via a target protein that is dynamically phosphorylated by cAMP-dependent protein kinase and dephosphorylated by protein phosphatase type 1. Mol. Reprod. Dev. 55:220-228, 2000.

Electrophysiological characterization of rat and mouse olfactory receptor neurons from an intact epithelial preparation

To understand the coding mechanisms underlying olfactory discrimination, it is necessary to characterize odor response properties of olfactory receptor neurons (ORNs). In contrast with rapid progress in molecular biology, there is little physiological data from ORNs in rodent. To facilitate acquisition of such data, we have developed an intact olfactory epithelial preparation from both rat and mouse. We have carried out initial studies of this preparation by monitoring odor responses by patch-clamping directly on the ORN dendritic knobs, a subcellular site very close to the locus of olfactory signal transduction. Our results show that rat and mouse ORNs have similar intrinsic membrane properties. Most cells fired spontaneously at a low frequency ( f) and about one half fired repetitively in response to current ( I) injection with a linear f/ I relation. About one third of rat and mouse ORNs responded to a mixture of four odors in a dose-dependent manner and about 60% of them responded to IBMX, a potent inhibitor of phosphodiesterase. The results suggest that this intact preparation offers the advantage of approximating in vivo physiological conditions, while furnishing an opportunity to map single neuron responses in the epithelium in a spatially-defined manner, using electrophysiological or cell imaging methods.

Methyl donor for 1-methyladenine biosynthesis in starfish ovarian follicle cells.

1-Methyladenine (1-MeAde), the oocyte maturation-inducing substance of starfish, is produced by ovarian follicle cells upon stimulation with a gonad-stimulating substance (GSS) released from radial nerves. It has been reported that a process of methylation is involved in GSS-induced 1-MeAde production by starfish ovarian follicle cells. The present study sought to identify a possible methyl donor for 1-MeAde biosynthesis in follicle cells of the starfish Asterina pectinifera. When isolated follicle cells were incubated with [methyl-(14)C]methionine (Met), there was an increase in the level of radiolabeled S-adenosylmethionine (SAM). After further incubation with GSS, the [methyl-(14)C]SAM level decreased, concomitant with a marked increase in radiolabeled 1-MeAde production. The amount of [methyl-(14)C]SAM consumed under the influence of GSS was similar to the amount of [methyl-(14)C]1-MeAde produced. Therefore, it is concluded that SAM is a methyl donor for 1-MeAde biosynthesis in starfish ovarian follicle cells. On the other hand, it is likely that the purine molecule of 1-MeAde is not derived from SAM but from ATP. 3-Isobutyl-1-methylxanthine, a potent inhibitor of cyclic AMP phosphodiesterase, also caused a reduction in the level of radiolabeled SAM following 1-MeAde production. This suggests that the methylation process of 1-MeAde biosynthesis in starfish ovarian follicle cells upon stimulation with GSS is mediated by a second messenger, cyclic AMP.

The cGMP-specific phosphodiesterase inhibitor E4021 dilates the pulmonary circulation.

We investigated the pulmonary vascular effects of E4021, a potent inhibitor of cGMP-specific phosphodiesterase, in control late-gestation fetal lambs, and in newborn lambs with persistent pulmonary hypertension (PPHN) after prenatal ligation of the ductus arteriosus. E4021 alone significantly relaxed fifth-generation pulmonary arteries isolated from control fetal lambs, an effect completely blocked after inhibition of nitric oxide synthase (NOS). In contrast, E4021 did not relax pulmonary arteries isolated from hypertensive lambs. Pretreatment with E4021 (10(-7) M) significantly enhanced relaxations to the NO donor S-nitrosyl-acetyl-penicilamine (SNAP) in arteries from both control and hypertensive lambs. In control, fully instrumented fetal lambs, infusions of E4021 (31 microgram/min) selectively dilated the pulmonary circulation, an effect again blocked after inhibition of NO synthase. Further studies were performed in newborn lambs with PPHN to study the vascular effects of E4021 alone, and in combination with inhaled NO. E4021 alone (1 to 100 microgram/kg/min) decreased pulmonary artery pressure (Ppa) in a dose-dependent fashion, and had minimal effect on systemic pressure. At the highest dose (100 microgram/kg/min), the dilation was selective for the pulmonary circulation. In subsequent protocols, E4021 (10 microgram/kg/min) significantly decreased Ppa and pulmonary vascular resistance (PVR), but these pulmonary vascular effects were not enhanced after NO inhalation at 0.5 or 5 ppm. We speculate that the lack of enhancement was due to the dramatic effects of E4021 alone. Potent, specific phosphodiesterase inhibitors such as E4021 may prove to be useful in the treatment of PPHN.

Methyl donor for 1‐methyladenine biosynthesis in starfish ovarian follicle cells

1-Methyladenine (1-MeAde), the oocyte maturation-inducing substance of starfish, is produced by ovarian follicle cells upon stimulation with a gonad-stimulating substance (GSS) released from radial nerves. It has been reported that a process of methylation is involved in GSS-induced 1-MeAde production by starfish ovarian follicle cells. The present study sought to identify a possible methyl donor for 1-MeAde biosynthesis in follicle cells of the starfish Asterina pectinifera. When isolated follicle cells were incubated with [methyl-14C]methionine (Met), there was an increase in the level of radiolabeled S-adenosylmethionine (SAM). After further incubation with GSS, the [methyl-14C]SAM level decreased, concomitant with a marked increase in radiolabeled 1-MeAde production. The amount of [methyl-14C]SAM consumed under the influence of GSS was similar to the amount of [methyl-14C]1-MeAde produced. Therefore, it is concluded that SAM is a methyl donor for 1-MeAde biosynthesis in starfish ovarian follicle cells. On the other hand, it is likely that the purine molecule of 1-MeAde is not derived from SAM but from ATP. 3-Isobutyl-1-methylxanthine, a potent inhibitor of cyclic AMP phosphodiesterase, also caused a reduction in the level of radiolabeled SAM following 1-MeAde production. This suggests that the methylation process of 1-MeAde biosynthesis in starfish ovarian follicle cells upon stimulation with GSS is mediated by a second messenger, cyclic AMP. Mol. Reprod. Dev. 54:63–68, 1999. © 1999 Wiley-Liss, Inc.

Solid phase parallel synthesis of highly substituted thiophene derivatives and identification of novel phosphodiesterase-4 (PDE-4) inhibitors

A versatile protocol for solid phase synthesis of highly substituted thiophene derivatives and their activity against the PDE-4 enzyme are discussed. This protocol employs 3-hydroxymethylthiophene-2-boronic acid ( 5) as the scaffold and sequential palladium catalyzed cross-coupling reactions as the CC bond forming step. This methodology allows convenient modification of the thiophene core from three directions, giving rise to structurally diverse derivatives with overall high chemical purity and yield. A novel series of potent PDE-4 inhibitors have been identified from these compounds.

Effect of cilostazol on the neuropathies of streptozotocin-induced diabetic rats.

ObjectivesThis study examined the effect of cilostazol, a potent phosphodiesterase inhibitor, on the progression of neuropathies associated with streptozotocininduced diabetes mellitus in Sprague-Dawley rats.MethodsEight weeks after streptozotocin treatment, a pelleted diet containing 0.03% cilostazol (15mg/kg body weight) was given for four weeks. Body weight, blood glucose level, motor nerve conduction velocity(MNCV), myelinated fiber density and size distribution of sciatic nerves were compared between age-matched normal rats (Group 1), control diabetic rats (Group 2) and cilostazol-treated diabetic rats (Group 3).ResultsBody weight was significantly reduced and blood glucose level was significantly increased in diabetic rats (Group 2 and 3) compared to normal rats. MNCV and cAMP content of sciatic nerves were significantly reduced in diabetic rats 12 weeks after streptozotocin treatment. Myelinated fiber size and density were also significantly reduced, and thickening of the capillary walls and duplication of the basement membranes of the endoneural vessels were observed in the diabetic rats. Whereas both body weight and blood glucose level of Group 3 did not differ significantly from those of Group 2, cilostazol treatment significantly increased MNCV and cAMP content of sciatic nerves in Group 3 but not to the levels observed in Group 1. MNCV positively correlated with cAMP content of sciatic nerves (r=0.86; p < 0.001). Cilostazol treatment not only restored myelinated fiber density and size distribution but reversed some of the vascular abnormalities.ConclusionThese findings suggest that a reduced cAMP content in motor nerves may be involved in the development of diabetic neuropathy, and that cilostazol may prevent the progression of diabetic neuropathy by restoring functional impairment and morphological changes of peripheral nerves.

Synthesis of 7-benzylamino-6-chloro-2-piperazino-4-pyrrolidinopteridine and novel derivatives free of positional isomers. Potent inhibitors of cAMP-specific phosphodiesterase and of malignant tumor cell growth.

7-Benzylamino-6-chloro-2-piperazino-4-pyrrolidinopteridine (7a) is a potent inhibitor of the cAMP-specific phosphodiesterase isoenzyme family PDE4 and induces growth inhibition in a panel of tumor cell lines. In this study, we describe a synthesis that yields 7a and novel derivatives free of positional isomers. The synthesis of alkylamino substituted pteridines is based on the successive nucleophilic aromatic substitution of the chlorine atoms of 2,4,6, 7-tetrachloropteridine. For the reaction with secondary amines, the positional order of reactivity was found to be C4 > C7 > C2 > C6. Final structural proof is given by X-ray crystallography. To unravel structural elements of 7a crucial for the interaction with the target enzyme, the compound was modified systematically. The impact of the modifications on activity was tested by evaluating the ability of the compounds to inhibit cAMP hydrolysis by cAMP-specific phosphodiesterase (PDE4) purified from the solid human large cell lung tumor xenograft LXFL529. Growth inhibitory properties were determined by in vitro treatment of the respective cell line LXFL529L using the sulforhodamine B assay (SRB). The results show that for high activity, the heterocyclic substituent in position 2 of the pteridine ring system requires the presence of a basic nitrogen in 4'-position, as represented by piperazine.

Thalidomide analogs and PDE4 inhibition

N-Phthaloyl 3-amino-3-arylpropionic acid analogs of thalidomide that are potent inhibitors of tumor necrosis factor-α are reported. These compounds were found to be potent inhibitors of phosphodiesterase 4.

ChemInform Abstract: 1,4‐Cyclohexanecarboxylates: Potent and Selective Inhibitors of Phosphodiesterase 4 for the Treatment of Asthma.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

EFFECTS OF SILDENAFIL ON THE RELAXATION OF HUMAN CORPUS CAVERNOSUM TISSUE IN VITRO AND ON THE ACTIVITIES OF CYCLIC NUCLEOTIDE PHOSPHODIESTERASE ISOZYMES

Purpose Sildenafil, an inhibitor of cGMP-specific phosphodiesterase 5 (PDE5), is currently undergoing evaluation as an oral therapy for penile erectile dysfunction. The aims of this study were to investigate the mechanism of action of sildenafil on the neurogenic relaxation of human corpus cavernosum (HCC) in vitro and to determine the activity of sildenafil against a full range of PDE isozymes. Materials and Methods Strips of HCC tissue were precontracted with phenylephrine. Relaxation responses resulting from electrical field stimulation (EFS) were then determined in the presence and absence of sildenafil. The effects of sildenafil on PDE1 to 5 prepared from human tissues and PDE6 from bovine retina were determined by measuring the conversion of [( 3) H]-cGMP or [( 3) H]-cAMP to their respective [( 3) H]-5′-mononucleotides. Results Sildenafil (0.001 to 1 micro M) enhanced the EFS-induced, nitric oxide (NO) dependent, relaxation of HCC in a concentration-dependent manner to a maximum of 3 times the pretreatment level at 1 micro M sildenafil. Compared with zaprinast, an early PDE5 inhibitor, sildenafil was approximately 240-fold more potent, inhibiting PDE5 from HCC with a geometric mean IC 50 of 3.5 nM. For sildenafil, IC 50 values for inhibition of PDE1 to 4 were 80 to more than 8500 times greater than that for PDE5 and the IC 50 for PDE6 (33 nM) was approximately 9-fold greater. Conclusions The data support the proposal that enhancement of penile erection by sildenafil in patients with erectile dysfunction involves potentiation of the NO-stimulated cGMP signal mediating relaxation of cavernosal smooth muscle during sexual stimulation. Sildenafil is a potent inhibitor of PDE5 from HCC, with high selectivity for PDE5 relative to other PDE isozymes.

Use of Cilostazol, a Novel Antiplatelet Agent, in a Post-Palmaz-Schatz Stenting Regimen

We evaluated the therapeutic efficacy of cilostazol, a novel potent inhibitor of phosphodiesterase, for the prevention of stent thrombosis following implantation of a Palmaz-Schatz stent guided by angiographic visual estimation alone in 71 patients with 84 lesions. Patients received 81 mg of aspirin 3 times daily and 100 mg of cilostazol twice daily after angiographic confirmation of optimal Palmaz-Schatz stent implantation. Of the 84 vessels stented, 65 (77%) were classified as type B2 or C lesions according to the modified American Heart Association/American College of Cardiology classification, and 51 (61%) were <3.0 mm in diameter. Multiple stents were used in 26 patients (31%). The final balloon inflation pressure was 18.3 ± 1.5 atm. The balloon-to-vessel ratio was 1.18 ± 0.16. No patient received heparin or warfarin after the procedure. There were no deaths, Q-wave myocardial infarctions, in- or out-of-hospital stent thrombosis, coronary bypass surgery, or serious side effects such as neutropenia and/or liver dysfunction during the 1-month follow-up period. These results indicate that cilostazol was a safe and effective antiplatelet agent with minimum side effects after Palmaz-Schatz stent implantation.We evaluated the usefulness of cilostazol, a novel antiplatelet agent, for the prevention of stent thrombosis. No deaths, Q-wave myocardial infarctions, stent thrombosis, or serious side effects such as neutropenia occurred during the 1-month follow-up period, which suggests cilostazol is a safe and effective antiplatelet agent after Palmaz-Schatz stent implantation.

Type 4 Phosphodiesterase Inhibitors Have Clinical and In Vitro Anti-inflammatory Effects in Atopic Dermatitis

Increased cyclic AMP-phosphodiesterase activity in peripheral blood leukocytes is associated with the immune and inflammatory hyperreactivity that characterizes atopic dermatitis. Atopic phosphodiesterase has high sensitivity to a variety of enzyme inhibitors, suggesting an increased therapeutic advantage. The objective of this study was to use in vitro assays to identify a potent phosphodiesterase inhibitor and then to investigate its effectiveness in treating atopic dermatitis. Leukocyte enzyme activity was measured by radioenzyme assay, whereas prostaglandin E2 and interleukins 10 (IL-10) and 4 (IL-4) were measured in 24-h culture supernatants of mononuclear leukocytes by immunoassays. The effect of a topical phosphodiesterase inhibitor on atopic dermatitis lesional skin was assessed by double-blind, paired comparisons of active drug and placebo ointments applied to symmetrically involved sites over a 28-d period. Using in vitro, assays, we demonstrated the ability of selective high-potency phosphodiesterase inhibitors to reduce prostaglandin E2, IL-10, and IL-4 production in atopic mononuclear leukocyte cultures. We selected the Type 4 phosphodiesterase inhibitor, CP80,633, based on its inhibitory potency, for clinical testing by topical, bilateral paired comparisons in 20 patients with atopic dermatitis and demonstrated significant reductions of all inflammatory parameters. Phosphodiesterase inhibitors modulate several pathways contributing to the exaggerated immune and inflammatory responses, which characterize atopic dermatitis. This in vivo demonstration of anti-inflammatory efficacy may provide a useful alternative to the over-reliance on corticosteroid therapy in atopic disease.

Decreased sensitivity to beta-adrenergic stimulation of the ventricular cells isolated from the spontaneously hypertensive rat heart.

1. The stimulatory effects of isoproterenol on the L-type Ca2+ current (ICa) were compared between the control (WKY) and hypertensive (SHR) rat heart cells, using the patch-clamp method. 2. The current density and the shape of the current-voltage relationship for ICa were not different between the two groups. However, the maximal percentage increase in response to isoproterenol was smaller in SHR (+91% in SHR and +81% in WKY), and the ED50 was significantly higher in SHR (0.081 mu mol/L in SHR and 0.020 mu mol/L in WKY). IBMX, a potent phosphodiesterase inhibitor, significantly increased the isoproterenol-stimulated ICa in SHR, but not in WKY. These results suggest an impaired cAMP production in SHR heart cells.

Design and synthesis of conformationally constrained analogues of 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one (Ro 20-1724) as potent inhibitors of cAMP-specific phosphodiesterase.

The synthesis and biological evaluation of cAMP-specific phosphodiesterase (PDE IV) inhibitors is described. The PDE IV inhibitor 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one (Ro 20-1724, 2) was used as a template from which to design a set of rigid oxazolidinones, imidazolidinones, and pyrrolizidinones that mimic Ro 20-1724 but differ in the orientation of the carbonyl group. The endo isomer of each of these heterocycles was more potent than the exo isomer in an enzyme inhibition assay and a cellular assay, which measured TNF alpha secretion from activated human peripheral blood monocytes (HPBM). Imidazolidinone 4a inhibited human PDE IV with a Ki of 27 nM and TNF alpha secretion from HPBM with an IC50 of 290 nM. By comparison, Ro 20-1724 is significantly less active in these assays with activities of 1930 and 1800nM, respectively.

Effect of cilostazol on experimental diabetic neuropathy in the rat.

Two proposed mechanisms of diabetic neuropathy are microvascular ischaemia and a reduction in Na,K-ATPase activity. We evaluated the effect of cilostazol, a drug that is both a potent phosphodiesterase inhibitor that normalizes nerve Na,K-AT-Pase and a vasodilator, on nerve blood flow (NBF) to determine whether it would improve experimental diabetic neuropathy. We examined whether epineurally applied cilostazol acted as a vasodilator on the peripheral nerve of normal and diabetic rats, and whether feeding the rats a cilostazol-supplemented diet could improve diabetic neuropathy. Cilostazol increased nerve blood flow (NBF) in a dose-dependent fashion with an EC50 of 10(-5.74) mol/l. Cilostazol also normalized NBF in experimental diabetic neuropathy with a 10(-4) mol/l local application on the sciatic nerve. In diabetic neuropathy, a cilostazol-supplemented diet improved both NBF and nerve conduction in a dose- and time-dependent fashion. Potential mechanisms of action of cilostazol on the nerve include its effect on NBF, Na, K-ATPase, and restoration of the thromboxane:prostacyclin ratio. Cilostazol may have potential in the treatment of diabetic neuropathy.

Suppression of eosinophil function by RP 73401, a potent and selective inhibitor of cyclic AMP-specific phosphodiesterase: comparison with rolipram.

1. We have investigated the inhibitory potency of RP 73401, a novel, highly selective and potent inhibitor of cyclic AMP-specific phosphodiesterase (PDE IV), against partially-purified PDE isoenzymes from smooth muscle and the particulate PDE IV from guinea-pig eosinophils. The inhibitory effects of RP 73401 on the generation of superoxide (.O2-), major basic protein (MBP) and eosinophil cationic protein (ECP) from guinea-pig eosinophils have also been studied. 2. RP 73401 potently inhibited partially-purified cyclic AMP-specific phosphodiesterase (PDE IV) from pig aortic smooth muscle (IC50 = 1.2 nM); it was similarly potent against the particulate PDE IV from guinea-pig peritoneal eosinophils (IC50 = 0.7 nM). It displayed at least a 19000 fold selectivity for PDE IV compared to its potencies against other PDE isoenzymes. Rolipram was approximately 2600 fold less potent than RP 73401 against pig aortic smooth muscle PDE IV (IC50 = 3162 nM) and about 250 times less potent against eosinophil PDE IV (IC50 = 186 nM). 3. Solubilization of the eosinophil particulate PDE IV increased the potency of rolipram 10 fold but did not markedly affect the potency of RP 73401. A similar (10 fold) increase in the PDE IV inhibitory potency of rolipram, but not RP 73401, was observed when eosinophil membranes were exposed to vanadate/glutathione complex (V/GSH). 4. Reverse transcription polymerase chain reaction (RT-PCR), using primer pairs designed against specific sequences in four distinct rat PDE IV subtype cDNA clones (PDE IVA-D), showed only mRNA for PDE IVD in guinea-pig eosinophils. PDE IVD was also the predominant subtype expressed in pig aortic smooth muscle cells. 5. RP 73401 (Kiapp = 0.4 nM) was 4 fold more potent than (+/-)-rolipram (Kiapp = 1.7 nM) in displacing[3H]-(+/-)-rolipram from guinea-pig brain membranes.6. In intact eosinophils, RP 73401 potentiated isoprenaline-induced cyclic AMP accumulation(EC50 = 79 nM). RP 73401 also inhibited leukotriene B4-induced generation of *02- (IC50 = 25 nM), and the release of major basic protein (ICo = 115 nM) and eosinophil cationic protein (IC50 = 7 nM). Rolipram was 3-14 times less potent than RP 73401.7. Thus RP 73401 is a very potent and selective PDE IV inhibitor which suppresses eosinophil function suggesting that it may be a useful agent for the treatment of inflammatory diseases such as asthma. The greatly different inhibitory potencies of rolipram against PDE IV from smooth muscle and eosinophils(in contrast to the invariable effects of RP 73401) are unlikely to be attributable to diverse PDE IV subtypes but suggest distinct interactions of the two inhibitors with the enzyme.

Clinical evaluation on combined administration of oral prostacyclin analogue beraprost and phosphodiesterase inhibitor cilostazol

Among various oral antiplatelets, a combination of a novel prostacyclin analogue beraprost (BPT) and a potent phosphodiesterase inhibitor cilostazol (CLZ) may result in untoward clinical effects due to possible synergistic elevation of intracellular cAMP (cyclic adenosine 3′,5′-monophosphate). Thereby, a clinical study of the combined administration of the two agents was attempted. Twelve healthy volunteers were assigned to take BPT/CLZ in the following schedule; BPT: 40 μg at day I and 120 μg t.i.d. from day 7 to 14, CLZ: 200 mg t.i.d. from day 3 to 14. At various time intervals, physical examination and blood collection for ex vivo platelet aggregation and determination of intraplatelet cAMP were performed. Throughout the observation period, no significant alteration in vital signs was observed. Seven out of 12 subjects experienced headache of a short duration accompanying facial flush in one and nausea in one, especially after ingestion of CLZ. All of these symptoms, probably caused by the vasodilating effect of the two agents, were of mild degree and no special treatment was required. Intraplatelet cAMP content was gradually but significantly increased to 9.84± 4.59 pmol per 10 9 platelets at day 14 in comparison with the initial value (6.87±2.25 pmol). The platelet aggregability was significantly suppressed at various time intervals but no additive or synergistic inhibitory effect by the combined administration was noted. In conclusion, the combined administration of BPT/CLZ is safe at doses used in the study, though the beneficial clinical effect of the combined administration has yet to be elucidated.

Determination of a novel and potent cyclic GMP phosphodiesterase inhibitor, 4-([3,4-(methylenedioxy)-benzyl]amino)-6,7,8-trimethoxyquinazoline, in dog plasma by high-performance liquid chromatography.

A simple and accurate method for determining levels of 4-([3,4-(methylenedioxy)benzyl]amino)-6,7,8-trimethoxyquinazoline, a novel cyclic GMP phosphodiesterase inhibitor, in dog plasma was developed. This method involves extraction with diethyl ether and reversed-phase high-performance liquid chromatography with ultraviolet detection. Regression analysis showed that the method was linear over the range 1.0-1000 ng/ml. The method was employed for the analysis of plasma samples in a preliminary pharmacokinetic study in beagle dogs.